Alexandre Ambrogelly1, Sarath Gundllapalli2,3,4, Stephanie C. Herring2,3,4, Carla Polycarpo2,3,4, Carina Frauer2,3,4, Dieter Söll2,3,4
1Department of Molecular Biophysics and Biochemistry and Chemistry, Yale University, New Haven, CT 06520-8114, USA.
2Department of Molecular Biophysics and Biochemistry, Yale University, P.O. Box 208114, 266 Whitney Avenue, New Haven, CT 06520-8114.
3Institute for Pharmacy and Molecular Biotechnology, University of Hei-delberg, D-69117 Heidelberg, Germany.
4Instituto de Bioquimica Medica, Universidade Federal do Rio de Janeiro, CEP 21941-901, Rio de Janeiro, Brazil.
Tóm tắt
Pyrrolysine (Pyl), the 22nd naturally encoded amino acid, gets acylated to its distinctive UAG suppressor tRNA
Pyl
by the cognate pyrrolysyl-tRNA synthetase (PylRS). Here we determine the RNA elements required for recognition and aminoacylation of tRNA
Pyl
in vivo
by using the Pyl analog
N
-ε-cyclopentyloxycarbonyl-
l
-lysine. Forty-two
Methanosarcina barkeri
tRNA
Pyl
variants were tested in
Escherichia coli
for suppression of the
lac
amber A24 mutation; then relevant tRNA
Pyl
mutants were selected to determine
in vivo
binding to
M. barkeri
PylRS in a yeast three-hybrid system and to measure
in vitro
tRNA
Pyl
aminoacylation. tRNA
Pyl
identity elements include the discriminator base, the first base pair of the acceptor stem, the T-stem base pair G51:C63, and the anticodon flanking nucleotides U33 and A37. Transplantation of the tRNA
Pyl
identity elements into the mitochondrial bovine tRNA
Ser
scaffold yielded chimeric tRNAs active both
in vitro
and
in vivo
. Because the anticodon is not important for PylRS recognition, a tRNA
Pyl
variant could be constructed that efficiently suppressed the
lac
opal U4 mutation in
E. coli
. These data suggest that tRNA
Pyl
variants may decode numerous codons and that tRNA
Pyl
:PylRS is a fine orthogonal tRNA:synthetase pair that facilitated the late addition of Pyl to the genetic code.
